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Gene expression analysis of clams Ruditapes philippinarum and Ruditapes decussatus following bacterial infection yields molecular insights into pathogen resistance and immunity.

Moreira R.1, Balseiro P. 1, Romero A. 1, Dios S. 1, Posada D. 2, Novoa B. 1, Figueras A. 1*

1 Instituto de Investigaciones Marinas (IIM). Consejo Superior de Investigaciones Científicas (CSIC). Eduardo Cabello 6, 36208 Vigo. Spain

2 Department of Biochemistry, Genetics and Immunology, Universidad de Vigo, Spain.

Corresponding address:

Antonio Figueras Instituto de Investigaciones Marinas Consejo Superior de Investigaciones Científicas (CSIC) Eduardo Cabello 6, 36208 Vigo Spain Telephone number: +34 986214462 Fax number: +34 986292762 e-mail: [email protected]

ABSTRACT

The carpet shell clam (Ruditapes decussatus) and Manila clam (Ruditapes philippinarum), which are cultured bivalve species with important commercial value, are affected by diseases that result in large economic losses. Because the molecular mechanism of the immune response of bivalves, especially clams, is scarce and fragmentary, we have examined all Expressed Sequence Tags (EST) resources available in public databases for these two species in order to increase our knowledge on related with the immune function in these animals. After automatic annotation and classification of the 3,784 non-annotated ESTs of R. decussatus and 4,607 of R. philippinarum found in GenBank, 424 ESTs of R. decussatus and 464 of R. philippinarum were found to be putatively involved in immune response. These were carefully reviewed and reannotated. As a result, 13 immune-related ESTs were selected and studied to compare the immune response of R. deccusatus and R. philippinarum following a Vibrio alginolyticus challenge. Quantitative PCR was performed, and the expression of each EST was determined. The results showed that, in R. philippinarum, the immune response seems to be faster than that in R. decussatus. Additionally, expression of NF-κB activating genes in R. decussatus did not seem to be sufficient to promote an immune response after Vibrio infection. R. philippinarum, however, was able to trigger and efficiently regulate the transcriptional activity of NF-κB, even when low expression values were reported.

KEYWORDS Ruditapes decussates, Ruditapes philippinarum, Vibrio alginolyticus, EST, Immune-related genes, expression, qPCR.

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1. INTRODUCTION

Due to its increasing commercial value, the culture of the carpet shell clam (Ruditapes decussatus) and Manila clam (Ruditapes philippinarum) has grown in recent years. However, diseases caused by a wide range of microorganisms are associated with large economic losses (Gómez-León et al., 2005). The majority of bacterial diseases are caused by members of the Vibrio genus (reviewed in Gestal et al., 2008 and Paillard et al., 2004). In particular, the pathogen used in this study, Vibrio alginolyticus, was associated with high mortality rates (up to 73%) of R. decussatus larvae and spat in 2001 and 2002 in a commercial hatchery in Spain (Gómez-León et al., 2005). Although there are no reliable data regarding mortalities on seabeds, it is known that R. philippinarum is more resistant to physical stress and pathogens than is R. decussatus (FAO, 2006; Tanguy et al., 2007). Although bivalves lack a specific immune system, the innate responses seem to be an efficient defense method, involving circulating cells and a large variety of molecular effectors (Canesi et al., 2004; Olafsen, 1995; Ordás et al., 2000a, 2000b; Tafalla et al., 2003). Information regarding bivalve immune-related genes remains very scarce and fragmentary despite recent advances. Most of the available data for bivalves were obtained on Eastern and Pacific oysters (Crassostreaa virginica and C. gigas) and mussels (Mytilus galloprovincialis) (Fleury et al., 2009; Gueguen et al., 2003; Pallavicini et al., 2008; Venier et al., 2009; Wang et al., 2010). Limited information is available on the R. decussatus and R. philippinarum immune genes. Gestal et al. (2007) identified several ESTs related to R. decussatus immunity by stimulating with a mixture of dead bacterial strains, and Prado-Alvarez et al. (2009a) described immune-related ESTs expressed by Perkinsus olseni-infected R. decussatus. Only a few transcripts encoded by genes putatively involved in the immune response of R. philippinarum against Perkinsus olseni have been reported (Kang et al., 2006). The European Marine Genomics Network has significantly increased the number of known ESTs from commercial marine mollusk species (Tanguy et al., 2007); however, most of the available resources are not annotated. In the present work, we have analyzed, to the best of our knowledge, all available R. decussatus and R. philippinarum ESTs.

The main goal of this work was to describe and classify immune-related transcripts from R. decussatus and R. philippinarum and also find differentially expressed genes after

- 3 - bacterial infection in the two species. To our knowledge, this is the first molecular study comparing the immune response of these two clam species to Vibrio alginolyticus (strain TA15) infection.

2. MATERIALS AND METHODS

2.1. Selection and identification of ESTs.

All non-annotated sequences from R. decussatus and R. philippinarum were downloaded from the GenBank NCBI database, converted into FASTA format and automatically screened with BLASTX (Altschul et al., 1990) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences with an e-value lower than 10e-5 were further annotated manually upon inspection of relevant similarities. After this, Blast2GO (Conesa et al., 2005) was used to apply terms (Ashburner et al., 2000) to sequences. Default values in Blast2GO were used to perform the analysis, and ontology level 3 was selected to prepare the level pie charts. Sequences were then analyzed with CAP3 (Huang and Madan, 1999) to find contigs, and nucleotide sequences were translated into protein and analyzed to find conserved domains with ExPASy-PROSITE (Gasteiger et al., 2003) and BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Further selection of genes was performed according to the following criteria: presence in the two clam species, importance in the immune system and quality of the sequences (complete proteins/domains and good interspecies alignment).

2.2. Experimental infection with V. alginolyticus

Clams R. decussatus and R. philippinarum were obtained from a commercial shellfish farm (Vigo, Galicia, Spain) and maintained in open circuit filtered seawater tanks at 15°C with aeration. They were fed daily with Phaeodactylum tricornutum and Isochrysis galbana. Prior to the experiments, clams were acclimatized to aquaria conditions for one week. Clams (n=45 for each species) were notched in the shell in the adjacent area of the anterior adductor and injected with 100 µl of Vibrio alginolyticus (108 UFC/ml in filtered seawater) to mimic an intravalvar infection. Controls (n=45 for each species) were

- 4 - injected with 100 µl of filtered seawater. After stimulation, clams were returned to the tanks and maintained at 15ºC until sampling at 3, 6 and 24 hours after challenge. Hemolymph (1 ml) was withdrawn from the adductor muscle of the clams with an insulin syringe. Hemolymph from 5 individuals was pooled and three biological replicates were taken at each sampling point. Hemolymph was centrifuged at 4°C at 2500 g for 15 minutes. The pellet was resuspended in 1 ml of Trizol (Invitrogen), and RNA was extracted following the manufacturer’s protocol. After RNA extraction, samples were treated with Turbo DNase free (Ambion) to eliminate DNA. Next, concentration and purity of RNA were measured using a NanoDrop ND1000 spectrophotometer, and 0.7 μg of each sample was used to obtain cDNA with SuperScript III Reverse Transcriptase (Invitrogen). The cDNA obtained was stored at -20°C until use.

2.3. Real-time quantitative PCR

Specific PCR primers (Table 1) were designed from the selected sequences using the primer3 program (Rozen and Skaletsky, 2000) according to qPCR restrictions. Oligo Analyzer 1.0.2 was used to check for dimer and hairpin formation. Efficiency of each primer pair was then analyzed with seven serial five-fold dilutions of cDNA of R. decussatus and R. philippinarum and calculated from the slope of the regression line of the quantification cycle versus the relative concentration of cDNA (Pfaffl, 2001). A melting curve analysis was also performed to verify that no primer dimers were amplified. If these conditions were not accomplished, new primer pairs were designed. Real-time quantitative PCR was performed in the 7300 Real Time PCR System (Applied Biosystems). One microliter of five-fold diluted cDNA template was mixed with 0.5 μl of each primer (10 μM) and 12.5 μl of SYBR green PCR master mix (Applied Biosystems) in a final volume of 25 μl. The standard cycling conditions were 95 ºC for 10 min, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. All reactions were performed as technical triplicates and an analysis of melting curves was performed in each reaction. The relative expression levels of the genes were normalized using 18S as a reference gene, which was constitutively expressed and not affected by the Vibrio challenge, following the Pfaffl method (Pfaffl, 2001).

2.4. Phylogenetic analysis

For each gene, homologous amino acid sequences of vertebrates and invertebrates were gathered and aligned (whole protein, domains or part of a domain) using the T-Coffee

- 5 - server (Notredame et al., 2000) in regular computation mode and using the t_coffee_msa multiple alignment method. Ambiguously aligned regions were filtered with G-Blocks (Castresana, 2000) choosing all the options for a less stringent selection. ALTER (Glez- Peña et al., 2010) was then used to convert filtered alignments into the Phylip format, and ProtTest 2.4 (Abascal et al., 2005) was applied to find the best fit model of protein evolution and the substitution model parameters to construct maximum likelihood phylogenetic trees in PhyML (Guindon, 2003); the remaining chosen options were the prespecified but the type of tree improvement, NNI & SPR was chosen for a most robust analysis, 1000 bootstrap replicates to assess phylogenetic confidence. The resulting trees were visualized and edited with FigTree 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/) and used to study the phylogenetic position of our sequences compared to other representative species. Branch length estimates were used to study a potential relationship between gene expression and amino acid replacement (Duret and Mouchiroud, 2000; Drummond, 2005).

2.5. Statistical analysis

Expression results were represented graphically as the mean ± the standard deviation of the biological replicates (n=3). Differences between time groups were considered significant at p≤0.05. Statistical analysis was performed with SPSS Statistics 17.0 through a one-way ANOVA test to know the effect of the time over expression. The relationship between expression level and branch length on the phylogenetic trees (Table 4) was studied using Kendall’s and Spearman’s non-parametric correlation analysis. Branch length was defined as the mean branch length from conspecific sequences to their most recent common ancestor.

3. RESULTS 3.4. EST selection

We were able to find 3,784 non-annotated ESTs of R. decussatus and 4,607 of R. philippinarum in GenBank (Figure 1). After removing sequences that belonged to unknown proteins and had e-values higher than 10e-05, the remaining ESTs that were related to the immune system were chosen; 424 ESTs of R. decussatus and 464 of R. philippinarum (table 2) were then manually revised.

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Gene ontology results showed that ESTs from both species followed the same distribution for the immune EST classification. Regarding cellular components (Figure 2, A and B), a high number of annotated ESTs (about 44%) were included in the group of cell part; membrane-bounded organelle proteins represented 25.42% and 26.47% of ESTs from R. decussatus and R. philippinarum, respectively; the third most represented group was the organelle part (15.25% and 10.29% of the sequences, respectively). Within molecular function classification (Figure 2, C and D), the most represented group in both species was protein binding activity (over 23.5% of sequences); catalytic activity was the other main group with a high number of ESTs involved in processes (14.18% of R. decussatus and 22.30% of R. philippinarum ESTs). Regarding biological process (Figure 2, E and F), metabolism was the highest represented group (approximately 36% of the sequences in both species); response to stimulus and stress was the second most represented group (9.14% of R. decussatus EST and a higher rate of 16.62% for R. philippinarum); cell communication and death processes were other important groups with 4-5% of the annotated ESTs each. From immunity-related ESTs, a final selection was performed according to presence in the two clam species, relevant role in immune response and high intraspecific homology among sequences belonging to the same gene. After this second selection step, the final number of ESTs was 51 for R. decussatus and 61 for R. philippinarum, corresponding to 13 genes (Table 3) that were grouped in several functional categories, as follows: receptors (host-pathogen interaction), including tandem repeat galectin, Toll-like receptor; heat shock proteins (22 and 40); genes related with complement (C1q domain- containing protein and thioester-containing protein); genes involved in apoptosis (inhibitor of apoptosis protein and APAF1-interacting protein homolog; cytokine-related molecules (LPS-induced TNF-alpha factor and inhibitor of nuclear factor kappa B); and others, such as ferritin, thrombin and high-mobility group 1 protein.

3.5. Expression analysis after V. alginolyticus challenge

Tandem repeat galectins (TRGal) and Toll-like receptors (TLR) function as pattern recognition receptors (PRR) and host-pathogen interacting molecules. The former is soluble and the latter membrane anchored. TRGal expression (Figure 3A) showed opposite expression patterns after infection of R. decussatus and R. philippinarum. In R. decussatus, the expression tended to diminish over time, whereas in R. philippinarum, the TRGal expression increased significantly over time. TLR also showed distinct expression

- 7 - patterns in the two species (Figure 3B): R. decussatus presented a high level of expression 6 h after challenge, whereas in R. philippinarum, the expression level increased through the time course, reaching the maximum at 24 h. The Inhibitor of Apoptosis Protein (IAP) and APAF-1 interacting protein (APIP) are inhibitors of apoptosis. The trends in IAP expression (Figure 3C) were similar in both species, with the maximum expression observed three hours after infection and a decrease afterwards. However, the magnitude of the decrease was different between both species. Although R. decussatus displayed an insignificant and gradually decreasing expression pattern, the diminution was especially evident in R. philippinarum, with significant values (p<0.05) with regard to 6 and 24 hours after infection. On the other hand, opposite trends between the two species were observed in the case of APIP expression (Figure 3D).

Another group of genes, including heat shock protein 22 (HSP22), ferritin and thrombin, was considered in this study for their likely role in the immune response and because they commonly function as activators of NF-κB. HSP22 is a small HSP that plays different roles as a molecular chaperone, ferritin captures circulating iron to overcome an infection and thrombin is involved in wound repairing and chemotaxis regulation in the affected area. The TLR previously described also participates in the signal transduction pathway leading to NF-κB activation. The expression profile of these four genes was similar in both species (Figure 3B, E, F and G). Whereas in R. decussatus the maximum expression occurred at 6 h to drop drastically at 24 h (only significant for HSP22 and TLR), R. philippinarum showed an increase in expression through the time course, with the maximum expression detected at 24 h (again significant for HSP22 and TLR). Figure 4 shows genes that had no detectable or very low expression in R. decussatus. The inhibitor of NF-κB (IκB) retains NF-κB in the cytoplasm through formation of a complex, which prevents translocation of NF-κB to the nucleus and thus blocks its subsequent function as transcription factor. IκB displayed a maximum expression at 24 h in the two species, but the fold change values were very different (1.7 in R. decussatus and 24.8 in R. philippinarum) (Figure 4A). Additionally, expression of IκB in R. decussatus was much lower than in R. philippinarum and remained undetectable until 24 post-infection.

The last group of studied genes (The LPS induced TNF-α factor, LITAF; the high mobility group 1 protein, HMG1; the heat shock protein 40, HSP40; the C1q containing

- 8 - domain protein; C1q and the thioester containing protein; thioester) showed a similar behavior (Figure 4B, C, D, E and F). The expression of this gene set in R. decussatus was undetectable in hemocytes at the sampling points considered. However, in R. philippinarum, their expression was higher. LITAF is a transcription factor that mediates TNF-α expression and other cytokines and genes implicated in immune response and apoptosis. HMG1 is a nuclear protein that can act as a cytokine in response to proinflammatory stimuli. HSP40 functions as a molecular chaperone. C1q and thioester are two proteins related to the complement system. The expression pattern in R. philippinarum showed a progressive increase over time following infection ultimately reaching the maximum at 24 h, except for thioester (Figure 4F), which follows an expression pattern defined by a high level of expression at 3 and 24 h and low expression at 6 h.

3.6. Phylogenetic analysis

The phylogenetic position of R. decussatus and R. philippinarum agreed with that expected, i.e, together with other mollusks, except for some cases in which the lack of information in the sequences resulted in alternative, unsupported relationships with low bootstrap values (thrombin, TLR, IAP and C1q). (Supplementary material, figures 1, 2 and 3). In general, in these gene trees R. philippinarum showed larger branches than R. decussatus, except for HMG1 and HSP40. However, we noticed that some loci with identical protein sequences for the two clam species could show different expression patterns (IκB). On the other hand, loci with different protein sequences for both species could display very similar expression patterns (IAP). In fact, we only found a statistically significant relationship between evolutionary change and expression level at the APIP locus.

4. DISCUSSION

Although the generation of ESTs of non-model organisms has been increasing in the last several years, this information frequently remains difficult to access. Identification and annotation of sequences is sometimes difficult, taking into account the important

- 9 - divergence among species and the lack of genetic information for many of them. Considerable efforts, however, have been made in some cases; for example, in the creation of a public EST database for Mytilus galloprovincialis (MytiBase) (Venier et al., 2009) and Crassostrea gigas (Fleury et al., 2009) and the further characterization of the EST collections (Venier et al., 2011). To our knowledge, this is the first comparative study of the expression of several immune-related genes between R. decussatus and R. philippinarum. It is also the first time that genes, such as IAP, APIP, thrombin or HMG1 are described in mollusks. We also report here new TLR expression data that have not previously been well described in this animal group (Qiu et al., 2007). The gene expression results after a Vibrio alginolyticus challenge showed that, for the genes and sampling points considered, R. decussatus presented lower expression levels than did R. philippinarum. In fact, some genes (IκB, LITAF, HMG1, C1q, thioester and HSP40) displayed very low or undetectable expression in R. decussatus hemocytes. These results coincided with previous studies performed in mollusks. It was described that the oyster HSP22 (Zhang et al., 2010) and scallop and abalone LITAF (Zhang et al., 2009; De Zoysa et al., 2010) were constitutively expressed in all studied tissues. However, hemolymph was one of the tissues with the lowest expression. Regarding thioester and C1q, it is known that, 3 hours after a Vibrio challenge, the expression level of C1q in hemocytes did not show a significant increase, compared to expression levels in the controls. The expression level of C1q increased rapidly 1 hour after a Vibrio challenge, but decayed 3 hours after infection (Gestal et al., 2010). This could explain why C1q also showed lower expression after 3 and 6 h in both clam species. In the case of thioester, Chlamys farreri showed no expression in hemocytes (Zhang et al., 2007). In Ruditapes decussatus hemocytes, expression of C3 (a thioester containing protein) was 6- fold to 12-fold lower than that in the digestive gland and decayed below the expression in the controls from 1 h to 6 h after a Vibrio challenge in hemocytes (Prado-Alvarez, 2009b). HMG1 protein, a highly abundant and ubiquitous non-histone nuclear protein, was undetectable by q-PCR in R. decussatus. However, HMG1 expression was detected in R. philippinarum hemocytes, especially 24 h after Vibrio challenge. In mammals, HMG1 showed variable expression, depending on the tissue and sampling points (Wang et al., 1999; Sass et al., 2002; Hagiwara et al., 2007). On the other hand, thermal injury, which it is known to increase mRNA levels of proinflammatory cytokines in various tissues,

- 10 - induced HMG1 expression after 24 h in rats (Fang et al., 2002), in agreement with the maximum expression of HMG1 detected at 24 h in R. philippinarum. Expression of IκB was similarly undetected at the initial times post-infection in R. decussatus. Nevertheless, a high expression of NF-κB activating genes (TLR (Takeuchi and Akira, 2010), HSP22 (Guo et al., 2009), ferritin (Ruddell et al., 2009) or thrombin (Anrather et al., 1997; Xue et al., 2009)) was detected 6 hours after challenge in R. decussatus. This could be evidence that in R. decussatus NF-κB was activated in response to the bacterial challenge between 6 and 24 h post-infection. This also seems to indicate that, although high levels of NF-κB activators were detected, a poor NF-κB response could be achieved, as the low expression of IκB at 24 h seems to suggest. It is well known that IκB is one of the most highly transcribed genes after NF-κB activation (Sun et al., 1993) and could be used as an indicator of NF-κB transcription factor activity. In R. philippinarum, we observed a low expression of TLR, HSP22, ferritin and thrombin, but there are indications that an effective immune response is being performed. IκB was highly expressed 24 h post-challenge, probably limiting the reaction generated through NF-κB activity. Moreover, qPCR assays showed that R. philippinarum TRGal, TLR, LITAF, HMG1, C1q, thioester and HSPs were highly expressed at 24 h, and all exhibited at least a 2-fold increase with respect to controls. The R. decussatus deficiency in IκB expression and the expression profiles of NF-κB related genes might explain its lower resistance to pathogens and stress. Based on the temporal expression pattern of TRGal, TLR and proteins related to complement system, R. philippinarum seems to detect pathogens earlier than did R. decussatus. The immune response to V. alginolyticus could then be initiated in a few hours, resulting in the negative regulation of the inflammatory response, as reported by the high expression of IκB at 24 h. In fact, it was previously observed in R. philippinarum that the highest TRGal mRNA expression was in the mantle and gill, while hemocytes presented the lowest expression. Additionally, TRGal expression increased 24 h after a Vibrio challenge (Kim et al., 2008), which is in agreement with our results. Concerning apoptosis related proteins APIP and IAP, we noticed that whereas V. alginolyticus seemed to have no effect on APIP expression (due to the high deviations registered among pools), it elicited a strong effect on early IAP expression, which could lead to the protection of hemocytes from apoptosis. Although IAP and APIP are both related to apoptosis inhibition, their activation against bacterial infection might be quite different, given the differences observed in the expression patterns to the same stimulus

- 11 - for these two genes. IAP was the only studied gene where a common expression pattern in R. decussatus and R. philippinarum was detected. This could be an indication of the importance of the apoptosis pathway in the immune response in bivalves, as it has recently been reported in Mytilus galloprovincialis (Romero et al., 2011). Indeed a survivin–XIAP complex participates directly in NF-κB activation (Altieri, 2010). AMP synthesis is also related to IAP because Drosophila iap2 null mutants fail to induce the synthesis of antimicrobial peptides and are highly susceptible to infection by Gram- negative bacteria (Huh et al., 2007). From these results we can conclude that R. decussatus and R. philippinarum presented very distinct responses to Vibrio alginolyticus both in their gene expression patterns and/or expression levels. This was especially evident in transcription factor genes, such as LITAF and NF-κB (using IκB as indicator of its expression). After a Vibrio infection R. decussatus expression of NF-κB activating genes seemed to be insufficient to promote an immune response, furthermore, R. decussatus did not express LITAF. However, even when these activating genes were lowly expressed in R. philippinarum, this clam seemed to be able to efficiently trigger NF-κB transcriptional activity and LITAF levels were clearly detected after challenge. With reference to phylogenetic analysis, it is known that the best predictor of the evolutionary rate of a protein is expression level (Cherry, 2010). Specifically, more highly expressed proteins tend to have lower evolutionary rates. But in this case, and although branch length were larger for R. philippinarum in almost all gene trees, we did not find a correlation between expression and amino acid changes in the sequence, but we should consider that some of these sequences are relatively short and may lack information. Only in one case, APIP, a negative correlation was observed: R. decussatus, the species with higher expression value had shorter branch length than R. philippinarum, consistent with the actual hypothesis. To our knowledge, the present work constitutes the first approach to understanding the molecular basis of the immune response of two different clam species, one potentially resistant against pathogens and adverse environmental conditions (R. philippinarum) and another more susceptible (R. decussatus) in wild breeding cultures. Based on the gene expression analysis, R. philippinarum response appeared to be more effective and faster than R. decussatus. Additionally, R. decussatus did not seem to express transcription factors that could initiate an inflammatory response. However, the expression pattern of

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IκB in R. philippinarum suggested a negative regulation of this inflammatory response 24 hours after infection. Further research will improve our understanding of the biological function of all these immune-related genes in clams. Longer sampling times, mortality studies, protein characterization and functional studies, among other assays, will enhance our comprehension of the immune system differences between R. decussatus and R. philippinarum. Additionally, the knowledge of the molecular mechanisms of the immune response could help to find molecular markers related to pathogen resistance.

ACKNOWLEDGEMENTS This work has been funded by the Spanish Ministerio de Ciencia e Innovación (MICINN) (AGL2008-05111). R.M. wishes to acknowledge additional funding from the Spanish MICINN through a FPI Spanish research grant (BES-2009-029765).

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FIGURE LEGENDS Figure 1. Scheme of the procedure followed to identify the clams ESTs. Figure 2. Classification of R. decussatus and R. philippinarum ESTs by Gene Ontology Terms. A and B: cellular component level 3. C and D: molecular function level 3. E and F: biological process level 3. Figure 3. Expression level of TRGal (A), TLR (B), IAP (C), APIP (D), HSP22 (E), ferritin (F) and thrombin (G) in hemocytes from R. decussatus (white bars) and R. philippinarum (black bars) at 3, 6 and 24 hours after V. alginolyticus challenge. All qPCR reactions were performed as technical triplicates and the expression level of analyzed genes was normalized using the 18S rRNA. Fold change units were then calculated by dividing the normalized expression values of hemocytes from infected clams by the normalized expression values of the controls. Each bar represents the mean and standard deviation of three biological replicates. The asterisks indicate statistically significant values (p<0.05) through the time course and for each species independently. Figure 4. Expression level of IκB (A), LITAF (B), HMG1 (C), C1q (D), thioester (E) and HSP40 (F) in hemocytes from R. decussatus (white bars) and R. philippinarum (black bars) at 3, 6 and 24 hours after V. alginolyticus challenge. All qPCR reactions were performed as technical triplicates, and the expression level of analyzed genes was normalized using the 18S rRNA. Fold change units were then calculated by dividing the normalized expression values of hemocytes from infected clams by the normalized expression values of the controls. Each bar represents the mean and standard deviation of thee biological replicates. The asterisks indicate statistically significant values (p<0.05) through the time course and for each species independently.

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Table 1. Primer sequences used in quantitative PCR.

Primer name Species Secuencia (5’→3’) Mollusk 18S- F Both species GTACAAAGGGCAGGGACGTA Mollusk 18S-R Both species CTCCTTCGTGCTAGGGATTG F-TRGAL Both species CCTAGAAGTGACTGTGCCTTTG R-TRGAL Both species CGTCACAGTGTTGTCCTTTACG F-HSP22-dec R.decussatus GCGGTAGCGTGTAGCGAAC R-HSP22-dec R.decussatus TCCTTAACCCAGTCCGAAAAC F-HSP22-phil R.philippinarum GCAGTCAATTCAAACCTGAAGA R-HSP22-phil R.philippinarum TCGATTGCGTTTGGAAGAG F- APIP-dec R.decussatus TCATCCCCGCACCATATTG R-APIP-dec R.decussatus CTTCACCCTGAACAAGCTCTTTC F-APIP-phil R.philippinarum TGTTGTGGTCAGGGACAGAG R-APIP-phil R.philippinarum TCCTCCGTCGATTTGTTTTC F-IAP-dec R.decussatus CGCTTATCGAGCCATGTAAGA R-IAP-dec R.decussatus AAGCCCGAATACGTCAAGG F-IAP-phil R.philippinarum TTCATTTGGCGAATGTGAAC R-IAP-phil R.philippinarum CGCACGAAAAACACATCACT F-INFKB Both species TTTATGTTTCGCAAGGAAGGA R-INFKB Both species AACCATTTTCAGCCAGCACT F-FERRITIN-dec R.decussatus AACGAGGTGGACGTGTTGTT R-FERRITIN-dec R.decussatus CGCCTGATTAACGGTTTTCT F-FERRITIN-phil R.philippinarum CTTCACAATGTTGCCTGTGG R-FERRITIN-phil R.philippinarum CCGTGTCCGCTTCCTAGAC F-THROM-dec R.decussatus ATCCGAACCCAAAGGAAACT R-THROM-dec R.decussatus CGGCAAGGATAAAATCATCG F-THROM-phil R.philippinarum GCCTCATTTGGTCGTGTGATT R-THROM-phil R.philippinarum GATCGTTGCCCATTGATTCAA F-TLR-dec R.decussatus GTTTTCGCACGAGAAAGCA R-TLR-dec R.decussatus CGTTCCAAGAAGGCAACAAT F-TLR-phil R.philippinarum AATGTTCTAGCGTTGACGAGAATG R-TLR-phil R.philippinarum CGGTATTTATTGTGGCGTTTAGG F-HSP40 Both species TGGTGAAGAGGGTTTGAAGAA R-HSP40 Both species CCAAAGAAATCCCGGAAAAC F-C1Q-dec R.decussatus CATGTGGCTTGACCTCCTTC R-C1Q-dec R.decussatus TTACGCAGCCTCAACATCAC F-C1Q-phil R.philippinarum TCTTCCCGAGGATACCACTG R-C1Q-phil R.philippinarum TTTCCAAGGAGGTCGTATCG Thioester Both species TTCATTCCCAGAACCTGGAC Thioester Both species GCCTCCGAGATCGTTATTTTC F-LITAF-dec R.decussatus TGCTGTCTGATTCCGTTCTG R-LITAF-dec R.decussatus ACTGGTCCCCACTTCCTCTC F-LITAF-phil R.philippinarum TGCTATTGTTGGATGCTGGA

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R-LITAF-phil R.philippinarum CCACACTGCTGGTGACAGTT F-HMG1-dec R.decussatus GAAGTCTCCCACCCAAAACA R-HMG1-dec R.decussatus CAGCAGTGGACGAGTTGAGA F-HMG1-phil R.philippinarum TGGAGGTGGTCGAGGAAAAC R-HMG1-phil R.philippinarum GGATGTCCCTGCCTTTGTGT

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Table 2. Immune system related ESTs (first selection criteria).

Category and gene identity. BlastX Species and number of EST LECTINS R.decussatus R.philippinarum tandem repeat galectin 6 12 galectin 4-like protein transcript variant - 3 Lectin-galC1 - 1 sialic acid binding lectin 10 15 putative contactin-associated protein 1 - Mannose receptor - 3 C-type lectin 1 - 2 C-type lectin 2 1 - C-type lectin 4 - 1 C-type lectin 8 1 - C-type lectin 9 - 1 C-type lectin A 1 4 C-type lectin - 2 putative salivary C-type lectin - 1 putative perlucin 5 1 - putative perlucin 4 1 1 perlucin-like protein (isoform A/B/C) - 5 HEAT SHOCK PROTEIN R.decussatus R.philippinarum heat shock protein 22 (isoform1/2) 6 7 heat shock protein 40 2 4 heat shock protein 60 1 - heat shock protein 70 2 7 heat shock protein 90 - 7 FtsJ homolog 1 (E. coli) 1 - COMPLEMENT & C1q-LIKE PROTEIN R.decussatus R.philippinarum C1q domain-containing protein 9 7 complement component C3 1 2 complement factor B-like protein 2 1 mantle gene 4 - 1 mantle gene 6 3 - adiponectin 1 - thioester-containing protein 3 6 EP protein precursor. Heavy metal-binding protein HIP 1 4 R.decussatus R.philippinarum 1 6

- 23 - cathepsin C - 1 cathepsin D - 1 - 1 cathepsin I 1 - 4 5 - 2 2 - APOPTOSIS R.decussatus R.philippinarum brain protein I3 8 - autophagy-related protein 3 1 - programmed cell death protein 1 1 death-associated protein - 2 B-cell translocation gene 1 1 3 c-myc binding protein 1 - similar to arrestin domain containing 3 2 - securin 1 - Inhibitor of apoptosis protein-IAP 8 12 BCL2-associatied athanogene (BAG) 1 2 BCL2/adenovirus E1B 19kD-interacting protein 1 1 - APAF1-interacting protein homolog (APIP) 3 1 caspase-1 1 - caspase-7 1 - caspase-8 1 - caspase-b - 1 separin protein - 1 cytochrome c 7 5 AND INHIBITORS R.decussatus R.philippinarum 4 9 similar to Human (serine protease) 1 - cysteine proteinase preproenzyme - 1 serine proteinase inhibitor - 2 inhibitor - 1 Kazal-type serine proteinase inhibitor 3 8 Hemocyte Kazal-type serine proteinase inhibitor - 1 metalloproteinase 1 1 Agrin precursor - 1 Cystatin-A2 - 1 Kunitz-like protease inhibitor - 1 signal peptide -

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CYTOKINE SIGNALING R.decussatus R.philippinarum Interferon-induced protein 2 - 1 Interferon-induced protein 27 - 1 Interferon-induced protein 44 7 3 Interferon-related developmental regulator 1 1 - LPS-induced TNF-alpha factor 1 4 Tumor necrosis factor-like protein - 1 inhibitor of nuclear factor kappaB 2 1 SPRY domain-containing SOCS box protein, putative 1 - interleukin enhancer binding factor 2, 45kDa 1 - IMMUNE DEFENSE AND HOST-PATHOGEN INTERACTION R.decussatus R.philippinarum lisozyme - 7 ferritin 7 2 Uromodulin - 1 peptidoglycan recognition protein (4/S2/S3) 3 - peptidoglycan recognition protein precursor - 1 beta-glucan recognition protein - 1 defensin - 2 hemolysin - 1 allograft inflammatory factor - 2 similar to HLA-B associated transcript 1 2 3 BAT1 homolog 1 - DEAD (Asp-Glu-Ala-Asp) box polypeptide 49 1 - similar to HLA-B associated transcript 3 1 saposin 5 - splicing variant form of ficolin A 1 - myticin C precursor 1 - macrophage migration inhibitory factor II 2 - coagulation factor IX precursor 1 1 coagulation factor VII 1 - thrombin 2 1 alpha macroglobulin 1 - CELL SURFACE RECEPTORS R.decussatus R.philippinarum G protein-coupled receptor 2 4 Notch 2 - 3 thrombospondin 1 - 3 scavenger receptor cysteine-rich protein type 12 precursor - 2 stabilin 2 - 1 Toll-like receptor TLR2.1 1 -

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Toll-like receptor (AGAP012385-PA) 1 1 SET protein 1 - LMPX of lamprey 1 - B-cell receptor associated protein-like protein 1 - angiopoietin (2/3/Y1/salivary secreted) 1 7 CELULAR ADHESION R.decussatus R.philippinarum fasciclin-like protein 3 - hemicentin 1 1 - OTHER R.decussatus R.philippinarum High mobility group 1 protein 1 3 metallothionein 3 - hematopoietic stem/progenitor cells protein-like 1 - c-Jun protein 1 1 similar to KLHL6 protein 1 - similar to lysosomal membrane glycoprotein-2 1 - Src family associated phosphoprotein 1 1 - similar to VNN3 protein 1 - RuvB-like 2-like protein - 1 T-cell immunoglobulin and mucin domain containing 2 - 1 Strumpellin - 1 EF-hand calcium binding domain 10 - 1 hemagglutinin and aggregation factor - 1 catalase - 1

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Table 3. Immune system related ESTs selected for expression studies (second selection criteria).

ESTs ESTs Functional category BLASTx R.decussatus R.philippinarum Tandem repeat galectin 6 12 Host-pathogen interaction Toll-like receptor (AGAP012385-PA) 1 1 Inhibitor of apoptosis protein 8 12 Apoptosis APAF1-interacting protein homolog 3 1 Heat shock protein 22 (isoform1/2) 6 7 Activators of nuclear Ferritin 7 2 factor kappa B Thrombin 2 1 Inhibitor of nuclear factor kappa B 2 1 Cytokine production LPS-induced TNF-alpha factor 1 4 High mobility group 1 protein 1 3 Heat shock proteins Heat shock protein 40 2 4 C1q domain-containing protein 9 7 Complement Thioester-containing protein 3 6

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Table 4. Mean of branch length until the nearest common ancestor of studied sequences from R. decussatus and R. philippinarum.

Gene Ruditapes decussatus Ruditapes philippinarum TRGal 0.0333 0.1173 TLR 0.6909 1.1502 IAP 0.0442 0.5947 APIP 0.0054 0.0413 HSP22 0.0328 0.1157 Ferritin 0.0095 0.0301 Thrombin 0.8159 1.7741 IkB 0.8057 0.8457 LITAF 0.242 0.5393 HMG1 0.6161 0.24405 HSP40 0.5769 0.5605 C1q 0.8139 1.1739 Thioester 0.0231 0.0832

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SUPPLEMENTARY FIGURES Supplementary figure 1. Protein maximum likelihood trees showing relationships between selected ESTs (sequences with accession number) and the amino acid sequences of several representative species. Bootstrap values calculated over 1000 replicates are shown next to the branches. Branches with a bootstrap value lower than 50% are shown collapsed. TRGal (A), TLR (B), IAP (C), APIP (D), HSP22 (E), Ferritin (F). Alignment length (a.l.), number of amino acids, is indicated under the tree name. Supplementary figure 2. Protein maximum likelihood trees showing relationships between selected ESTs (sequences with accession number) and the amino acid sequences of several representative species. Bootstrap values calculated over 1000 replicates are shown next to the branches. Thrombin (A), IκB (B), LITAF (C), HMG1 (D). Alignment length (a.l.), number of amino acids, is indicated under the tree name. Supplementary figure 3. Protein maximum likelihood trees showing relationships between selected ESTs (sequences with accession number) and the amino acid sequences of several representative species. Bootstrap values calculated over 1000 replicates are shown next to the branches. Branches with a bootstrap value lower than 50% are shown collapsed. HSP40 (A), C1q (B), Thioester (C). Alignment length (a.l.), number of amino acids, is indicated under the tree name.

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Figure(s)

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Supplementary Material for online publication Click here to download Supplementary Material for online publication: Supplementary material.docx